Recombinase mediated DNA therapies

ABSTRACT

Nucleoproteins comprising overlapping homologous DNA sequences coated with a recombinase are employed for introduction into cells to produce double D-loop structures. The sequences may be modified with various moieties which result in modification of DNA. Formation of the D-loops results in inhibition of replication and transcription, where the moieties can be employed for permanent scission or modification of the DNA. The methods and compositions can be used for investigating physiological processes, for producing animal models, and for inhibition of an undesirable phenotype in vivo.

This application is a continuation of Ser. No. 08/381,674, filed Jan.31, 1995, now abandoned.

TECHNICAL FIELD

The field of this invention is the inactivation of target DNA in vivo.

BACKGROUND

The ability to selectively inhibit the growth of a subset of cells in amixture of cells has many applications in culture and in vivo. Where twosets of cells have distinguishing characteristics, such as tumor cellswhich require expression of one or more genes, which are not expressedin normal cells or only expressed at a low level, there is substantialinterest in being able to selectively inhibit the proliferation of thetumor cells. Where groups of cells are differentiating, and at one levelof differentiation, expression of a particular gene is required, theability to inhibit the expression of that gene can be of interest. Wherecells are infected by viruses, parasite or mycoplasmas, the selectiveability to inhibit the growth of the virus or mycoplasma can be animportant goal.

In the studies of metabolic processes, differentiation, activation, andthe like, there are many situations where it is desirable to be able toselectively inhibit the transcription of a particular gene. In this way,one can study the effect of a reduction in the transcription of the geneand expression of the gene on the phenotype of the cell. In theextensive efforts to understand embryonic and fetal development, todefine segmental polarity genes and their function, there is alsointerest in being able to selectively inhibit particular genes duringvarious phases of the development of the fetus.

As in the case of the studies in culture, selective inhibition ofparticular genes can also be of interest in vivo. In many situations,cellular proliferation can be injurious to the host. The proliferationcan be as a result of neoplasia, inflammation, or other process whereincreased number of cells has an adverse effect upon the health of thehost.

There is, therefore, substantial interest in finding techniques andreagents which allow for selective inhibition of particular genes, so asto control intracellular molecular processes.

Relevant Literature

WO93/05178 provides an extensive description of double D-loop formation,with an extended bibliography of references. Sena and Zarling, (1993)Nature Genetics 3:365-372 and Revet, Sena and Zarling, J. Mol.Biol.232:779-791 describe double D-loop formation. Golub et al., (1992)Nucleic Acids Res. 20:3121-5; Golub et al., (1993) Proc. Natl. Acad.Sci. USA. 90:7186-90; Ferrin and Camerini-Otero, (1991) Science254:1494-7.; Koob et al. (1992) Nucleic Acids Res. 20:5831-6; Revet etal. (1993) J. Mol. Biol. 232:779-91; Sena (1993) Nature Genetics3:365-371 and Jayasena and Jonnston (1993) J. Molec Biol. 230:1015-1024.

SUMMARY OF THE INVENTION

Recombinase coated pairs of single-stranded probes having a region ofcomplementarity are introduced into cells. One or both probes may bemodified with an agent capable of inhibiting transcription. The pairs ofrecombinase coated probes are introduced into cells, where the probesare directed to a DNA target sequence, particularly genes, primarilydirected to the 5'-transcriptional initiation region or other essentialtranscriptional initiation sequence, such as an enhancer. The formationof the double D-loop inhibits copying, e.g. transcription, and byproviding for a DNA modifying agent bound to one or both of the probes,further inhibition can be achieved.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Methods and compositions are provided for inhibiting copying of a DNAsequence, where copying intends transcription and replication. Ofparticular interest is selectively inhibiting the transcription of atleast one gene in a cellular host. The method comprises introducing apair of probes having a complementary region, where the pair of probesis coated with a recombinase. One or both of the probes may be furthermodified with an agent which can react with DNA to further inhibitcopying. The probes are introduced into the cells by any convenientmeans, in culture or in vivo. As a result of the introduction of theprobes into the cell, with formation of double D-loops, copying of thetarget DNA is substantially diminished, resulting in a change in thephenotype of the cells or mortality. The subject compositions may beused by themselves or in conjunction with other agents, depending uponthe purpose for which the subject compositions are employed.

The probes may be any sequences which have substantial homology witheach other and with a target DNA sequence. Usually the homology betweenthe two probes and the target sequence will be at least about 70%between target and probe, more usually at least about 90% and preferably100% (complementarity). By homology is intended sequences havingsubstantial identity and the percent homology is defined in accordancewith FASTP (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85, 2444-8(1988).

For the most part, the DNA sequences will be the naturally occurringnucleotides, although some modifications may be made, where a proportionof the nucleotides may be modified to enhance stability. Thus, variousoxygens of the phosphate in the backbone may be substituted with sulfur,nitrogen or carbon (methylene), or an unnatural sugar may be employed,e.g. replacing ribose with arabinose. To enhance stability, one or bothtermini of the sequence may be modified, particularly byfunctionalization. The modification may serve to inhibit exonuclease, tolink to another moiety, or the like. Thus, ethers, amino groups, esters,or other functionality may be provided at one or both termini. Reactivemoieties which affect the ability for copying may include singlestranded or double stranded DNA scission inducing molecules,intercalating molecules, cross linking molecules, photoactive molecules,and the like. Scission inducing molecules provide for cleavage of one orboth of the DNA strands of the target sequence. These may includechelated metal ions, such as iron and copper. Chelating agents mayinclude ethylenediamine tetraacetic acid (EDTA), nitrilo triacetic acid(NTA), 2,9-dimethyl-1,10-phenanthroline, etc. Alternative molecules mayinclude electron accepting molecules, such as quinones, and the like.

Depending upon the particular purpose of the subject composition,various agents which provide for cross linking may be employed.Photoactivatable cross linkers include coumarin, psoralen, and othercommercial cross-linking molecules available from Pierce and Pharmacia.Other compounds include oxetanes, or other unstable dioxacycliccompounds.

A large variety of intercalating agents are known, including many dyes,such as thiazole orange, ethidium bromide, phenanthridines, actinomycinD, etc. By intercalating into the DNA, these molecules will furtherstabilize the complex and reactivity and if chemically modified caninteract with the DNA strands to form covalent bonds.

In addition, one can provide for compounds which are activatedintracellularly to react with nucleotides. These compounds includequinones having various substituents.

The various active moieties may be tethered to the DNA sequence by abond or any convenient linking group, which may be a single atom or achain of about fifty atoms, where the atoms may include carbon,nitrogen, oxygen, sulfur, phosphorous, and the like. Thus, the chain maybe an oligonucleotide chain, peptide nucleic acid chain (nucleic acidchain, where phosphate is substituted with glycine), oligosaccharide,polyoxyalkylene, polyamide, e.g. polypeptide, alkylene, arylene,combinations thereof, or the like. The particular linking group is notcritical, but one may be selected over another for syntheticconvenience, in relation to the particular moiety, to providesolubility, flexibility, hydrophobicity, enhanced binding to DNA, or toremove secondary structure. Usually, there will be from 0 to 3 activemoieties per probe, more usually from 0 to 2, particularly 0 to 2 forthe combination of probes, more usually 0 to 1 for the combination ofprobes. The size of the probes, extent of homology, and areas ofnon-homology may vary widely. Thus, one may employ combinations ofpartially overlapping probes which alternate where the first pair hasthe 3' portion of one probe overlapping with the 5' portion of the otherprobe followed by the 3' portion available in the first pair of probesoverlapping with the 5' portion of the next probe and so on.Alternatively, one could have one long probe and three smallerhomologous probes. One could have a single pair of probes or acombination of two or more pairs of probes. The ssDNA probes may also beused, where the probes are conveniently proximal to the termini oflinear ssDNA, where the termini will be at least partially overlappingwhen hybridized to the target or contiguous when bound to the target. Ineffect, a single probe may be divided into two regions, which when thetwo regions are bound to the target sequence, the probes will besubstantially contiguous, e.g. spaced apart by fewer than about 3nucleotides. Thus one could have a single molecule comprising the twoprobes or two molecules, where the termini of the two molecules comprisea sequence which is complementary to the termini of a second moleculeand homologous to one strand of the target sequence. The probes orregions may be linked at either of their termini, preferably the 5'terminus of one being linked to the 3' terminus of the other, so as toform a circle when bound to the target, so that the 3' terminalnucleotide of one region is contiguous to the 5' terminal nucleotide ofthe other region in the 5'-3' direction of the ssDNA. Where the terminiare contiguous when bound to the target sequence, the ends may besubject to joining, including ligation, to prevent removal from thetarget sequence. For joining chemically, one or both of the contiguousends may be modified to provide an agent capable of chemical linkage.

The particular choice will depend upon the target sequence, thedifference between the target sequences and other sequences in the host,the desired stability of the D-loop, whether other moieties are presentfor stabilization, the particular target, and the like. There will be acombination of at least 2 probes having at least 30 nucleotide overlapswith at least 70% homology, usually at least 50 nucleotide overlap,preferably at least about 75 nucleotide overlap or greater. The probeswill be at least 30 nucleotides, preferably at least 50 nucleotides,more preferably at least 100 nucleotides, and may be 1000 nucleotides orgreater, usually being not more than about 500 nucleotides.

For the purposes of this invention, recombinases will be recA-likeproteins. These proteins may be characterized by forming a filament withssDNA, induces strand exchange in providing natural strand displacementby the incoming strand, and is normally associated with DNA repair. Inthe subject invention, the recombinase may be any protein which enhancesthe formation of the D-loops, by itself or in conjunction with othersubunits of a recombinase complex. Various naturally occurring or mutantrecombinases are available, particularly recA from E. coli, e.g.recA-803,Rad 51 and Rad 52, from S. cerevisiae, Rad51 like, DMC1, mei3from N. crassa, human recombinase from human cells, and the like. Seeparticularly page 15, lines 10-30 of WO93/05178. The ratio of therecombinase to the nucleotides of the probe will generally be in therange of about 1:3-50, more usually 1:3-25, depending upon the size ofthe probe, the smaller probes requiring higher levels of recombinase.The probes can be synthesized in accordance with conventionaltechniques, either manually or by automated synthesizers. By appropriatechoice of the terminal nucleotide, the terminal nucleotide may befunctionalized, so as to be resistant to exonuclease, provide for a sitefor linkage of the DNA deactivating moiety, and the like. For automatedsynthesizers, usually a 3' terminus will be linked by a cleavablefunctionality to a bead. This functionality may be employed to serve asthe 3' terminus and may also serve as a site for linkage of the DNAdeactivating moiety. The particular manner in which the subject probesare synthesized is not critical to this invention.

For coating the probes with the recombinase to produce the nucleoproteinfilament probe, one combines single-stranded DNA with the recombinase inan appropriate buffered medium in the presence of a cofactor andmagnesium ion. A variety of cofactors may be employed, such as ATPγS,rATP, dATP, GTPγS, or equivalent molecules, mixtures thereof, and thelike. The ratio of the recombinase per nucleotide of the probe willgenerally range from about 1-50:1 usually in the range of 2-6:1 moreusually in the range of 2-4:1. Thus, a high concentration of recombinaseand cofactor may be employed to provide for a substantially completecoating of the probe and to prevent reannealing of the ssDNA probes.Alternatively, lower amounts of the recombinase may be employed where alower ratio of recombinase to nucleotide will suffice. Generally, thecofactor will be in the range of about 0.2-12 mM, preferably about 2.4-8mM. The magnesium ion will generally range from about 4-25 mM, moreusually 6-8 mM, and may be conveniently present as the acetate. Theprobe may then be isolated and purified by any convenient means.Conveniently, the probes may be isolated by filtration, where the filterwill retain the recombinase coated probes, while allowing individualmolecules to pass through the filter, by gravity sedimentation,centrifugation, or the like. The probes may then be stored for use,particularly cold or frozen, where the pairs of probes may be combinedand retained in the same container. Conveniently, the probes may belyophilized and maintained as a dry powder, being reconstituted whenneeded.

For the most part, the subject invention will be used with a gene as thetarget sequence. However, the probes may be directed against any targetsequence associated with replication, such as a centromere, telomere,replication origin, or the like, repetitive sequences, etc. For targetgenes, the probes may be complementary to any gene of interest(including the 5' transcription regulatory region, which includes the 5'untransalated region, the coding region, and the transcriptionaltermination region), particularly the sequences associated withtranscription initiation, which include the TATA box, CAAT sequence, theassociated regions, enhancers, or other sequence essential totranscription. Therefore, the sequences specific for particulartranscription factors will also provide for targets. Alternatively, thesequences may be present in the coding region, at splice sites, or thelike. Combinations of probes may be employed, where the probes aredirected both to transcriptional elements and to coding sequences. Sincein each instance, one will achieve varying degrees of inhibition oftranscription and expression, for each target, one may optimize theparticular sequence. However, by directing the probes to the TATA-CAATregion, enhancer regions, and transcription factor binding sequences forresponse elements, one can substantially ensure the substantialinhibition of transcription of the gene.

Genes of interest will be any of a large variety of genes associatedwith housekeeping, proliferation, differentiation, activation,transcription, oncogenesis, and the like, where the genes will beassociated with cellular genes. Alternatively, the genes may be genesassociated with pathogens, including microorganism, parasitic and viralgenes, where a wide variety of genes may be targets, such as genesassociated with transcription factors, polymerases, reversetranscriptases, helicases, topoisomerases, capsid antigens, coatproteins, integrases, and the like.

The particular target can depend upon the particular purpose for whichthe probe is employed. Targets of interest include oncogenes,transcription factor genes, proliferation repressor genes, mutant tumorsuppressor genes, segmental polarization genes, homeobox genes,addressin genes, homing receptor genes, major histocompatibility complexgenes, immunoglobulin genes, cytokine genes, immunosuppressivetransforming growth factor genes, colony stimulating factor genes, drugpump genes (mdr genes), integrin genes, enzyme genes, cytostructuralgenes, membrane channel genes, etc. In some instances, one may wish toblock the 3' untranslated region (3' UTR), where the 3' UTR is known tohave a regulatory function. In this manner, one may determine whatfunctions are regulated by the 3' UTR. Target oncogenes for thetreatment of cancer include src, ras, sis, fos, erb, erbb2, neu, myc,gli, etc. Other genes to be inhibited include receptors, such as the EGFreceptor, estrogen receptors, PDGF receptor, viral receptors, includingCD4 for HIV, and the like.

Also, various specialized proteins may be of interest for inhibition,such as telomerases, in understanding senescence, heat shock proteins,in understanding response to adverse conditions in their activity inhelping folding of proteins, recombinases, in understanding processesinvolved with correction and DNA modification, viral integrases and repproteins in understanding processes in viral replication andintegration, polymerases, in understanding the roles specializedpolymerases play, zinc finger DNA binding proteins involved intranscription, and the like.

Pathogenic prokaryotes of interest include Vibrio, e.g. V. cholerae;Escherichia, e.g. Enterotoxigenic E. coli, Shigella, e.g. S.dysenteriae; Salmonella, e.g. S. typhi; Mycobacterium e.g. M.tuberculosis, M. leprae; Clostridium, e.g. C. botulinum, C. tetani, C.difficile, C.perfringens; Cornyebacterium, e.g. C. diphtheriae;Streptococcus, S. pyogenes, S. pneumoniae; Staphylococcus, e.g. S.aureus; Haemophilus, e.g. H. influenzae; Neisseria, e.g. N.meningitidis, N. gonorrhoeae; Yersinia, e.g. G. lambliaY. pestis,Pseudomonas, e.g. P. aeruginosa, P. putida; Chlamydia, e.g. C.trachomatis; Bordetella, e.g. B. pertussis; Treponema, e.g. T.palladium; and the like.

Pathogenic eukaryotes of interest include Cryptococcus, e.g. C.neoformans; Candida, e.g. C. albicans; Histoplasma, e.g. H. capsulatum;Coccidoides, e.g. C. immitus; Giardia, e.g.G. lamblia; Plasmodium, e.g.P. falciparum, P. malariae, P. vivax; Toxoplasma, e.g. T. gondii;Leishmania, e.g. L. mexicana; and the like.

Viral groups of interest include orthomyxoviruses, e.g. influenza virus;paramyxoviruses, e.g respiratory syncytial virs, mumps virus, measlesvirus; adenoviruses; rhinoviruses; coronaviruses; reoviruses;togaviruses, e.g. rubella virus; parvoviruses; poxviruses, e.g. variolavirus, vaccinia virus; enteroviruses, e.g. poliovirus, coxsackievirus;hepatitis viruses, e.e. hepatitis B virus, hepatitis C virus;herpesviruses, e.g. Herpes simplex virus, varicella-zoster virus,cytomegalovirus, Epstein-Barr virus; rotaviruses; Norwalk viruses;hantavirus; arenavirus, rhabdovirus, e.g. rabies virus; retroviruses,such as HIV, HTLV-I and -II; papovaviruses, e.g. papillomavirus;polyomaviruses; picornaviruses; and the like.

When the subject probes are used in culture, the probes will beintroduced into the culture at an effective concentration based on thenumber of cells to provide the desired level of inhibition. Usually, theratio of probe to target sequence will be in the range of about 1-30:1,more usually in the range of about 2-25:1. Therefore, the amount ofprobe which is employed will be dependent upon the number of targetsequences present, by virtue of the number of cells, the number ofcopies of the target sequence, the number of integrated viruses, thenumber of viral molecules, the number of episomal elements, or the like.The probes are able to cross the membrane barrier and be taken up by thecells, although various techniques can be employed to enhance theefficiency of translocation into the cytoplasm of the cell. For example,one may use liposomes, where the liposome comprises the fusogenic HVJprotein of the Sendai virus or respiratory syncytial virus or gramicidinS peptide. By providing for preparation of the liposomes in the presenceof the probes, the probes will be incorporated into the lumen of theliposomes. The liposomes will then fuse with the cellular membranereleasing the probes into the cytoplasm of the cell. Lipofection may beemployed using DOTAP (Boehringer Mannheim). Other techniques includeelectroporation, fusion, microinjection, biolistics, polyamidoaminedendrimer complexes, and the like.

The subject probes may also be used in mammalian hosts. Mammalian hostsinclude primates, particularly humans, domestic animals, such as bovine,equine, feline, canine, porcine, ovine, lagomorpha, murine, etc. Thesevarious hosts may be used for research purposes, for treatment ofindications associated with genetic disorders, for the treatment ofpathogens, and the like.

The subject compositions may be administered systemically or locally.For many applications, local administration will be preferred. Systemicapplication will generally involve parenteral application, particularlyinjection, where the injection may be intravascular, intramuscular,peritoneal, subcutaneous, etc. As indicated above, the subjectcompositions may be administered without incorporation into a liposomeor other vehicle or by incorporation into a liposome. Physiologicallyacceptable vehicles will be employed, such as water, saline, phosphatebuffered saline, ethanol, vegetable oil, etc. The amount of the probeswhich is employed will vary depending upon the particular target, themanner of administration, the frequency of administration, the stabilityof the probes, and the like. Generally, amounts which will be employedsystemically will provide for a blood concentration in the range ofabout 1 nM to 10 μM.

For local administration, various techniques may be employed.Particularly, for a region which can be reached with a needle, one mayuse the subject compositions in conjunction with a matrix which slowsthe transport of the subject compositions away from the locale at whichthe subject compositions are introduced, or with a pump which providesfor continuous local infusion. Various matrices have been employed, suchas collagen, fibrinogen, hyaluronic acid and the like. Generally, thesubject compositions will range in from about 0.5 to 70, more usuallyfrom about 1 to 35 weight percent of the composition. Other compositionsmay be present, such as vasoconstrictors, stabilizers, or other agents,depending upon the purpose for which the subject compositions areemployed.

For treatment of cancer, the subject compositions may be used inconjunction with cytotoxic agents, where the cytotoxic agents are at orbelow their normal concentration. Thus, by employing a combination ofthe subject compositions with cytotoxic agents, the cytotoxic agent canbe used at from about 10 to 60% of its normal therapeutic dosage.Cytotoxic agents include cis-plat, vinca alkaloids, 5-fluorouracil,adriamycin, methotrexate, actinomycin D, BCNU, etc.

The subject compositions may be used for inhibiting specific celllineage development, e.g., NK, LAK, B- and T-cell development, byinhibiting the expression of CD4, CD8, or a member of the CD3 complex.Other proteins associated with activation may also be the subject ofinhibition, either individually or in conjunction with the inhibition ofother genes. In addition, the subject compositions can be used toinhibit cytokines associated with specific activation, such as IL-2 andIL-4. By inhibiting expression of IL-4, allergic responses can bediminished. The subject compositions may also be employed in producinganimal models for a wide variety of diseases associated with geneticdefects. Thus, those diseases where the lack of a competent proteinresults in an adverse phenotype can be studied in animal models, whereby employing the subject compositions, expression of the particularprotein may be inhibited for an extended period of time. Also, byvarying the nature of the sequence, as to its terminal groups and degreeof homology, the period of time for the inhibition, as well as the levelof inhibition, may be modulated, so as to have a model where thephenotype may be reversed. Animal models may be developed associatedwith the inhibition of expression of apolipoproteins, cytokines,recombinases, proteins associated with differentiation, growth andmaturation, such as CD4, CD8, growth factor receptors, interferonreceptors, virus receptors, and the like. Particularly, mice and ratsmay be temporarily or permanently modified as to phenotype, dependingupon the nature of the probes, the concentration employed, whether theprobes have the ability to permanently modify the DNA, and the like.

The subject compositions can be provided as kits, where thecomplementary sequences uncoated or pre-coated with the recombinase andcomprising the side chain, as appropriate, can be supplied. Thus, atleast one pair of probes would be provided, conveniently in a singlevessel. Other combinations of probes may also be provided, either in thesame or different vessels, depending upon the nature of the target. Theprobes will normally be made available as a dry powder, which can bereconstituted prior to use. When used in vivo, the compositions will besterilized and maintained in a sterile container prior to use.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

I. Inactivation of c-Ki-ras protooncogene.

Two compositions were prepared comprising the sequence immediatelydownstream of the transcriptional start sites of the c-Ki-rasprotooncogene. (Hoffman, et al. (1990) Proc. Natl. Acad. Sci. USA 87:2705-2709; Barbacid (1987) Annu. Rev. Biochem. 86: 779-827).

In the first composition, coumarin is present at the 5' terminus byadding as the last nucleotide 3' -O-(N-diisopropylamino)phosphoramiditederivative of 4,4'-dimethoxytrityl derivative at the 5' position of the2'-deoxyriboside of 7-hydroxymethylcoumarin, followed by deprotection ofthe deoxyriboside. See U.S. Pat. No. 5,082,934. The coumarin substitutedoligonucleotide is then isolated in accordance with conventional ways.

The second composition has an DTPA-Fe chelate attached at the end of theoligonucleotide prepared in accordance with conventional ways. Theoligonucleotide was amino linked and coupled todiethylenetriaminepentaacetic acid (DTPA) isothiocyanate as previouslydesribed. (Dewanjee et al., Biotechniques 16:844-6 (1994)

The Y1 cell line, established from the mouse adrenocortical tumor LAF,was employed. (Schwab et al. (1983) Nature 303:497-501) Y1 has thec-Ki-ras protooncogene amplified 50 times.

With each of the above compositions, the following studies are carriedout:

10⁶ Y1 cells are seeded in plates the night before transfection in DMEMwith 10% FCS and maintained under normal growth conditions. The coatedoligonucleotide compositions are prepared by coating the aboveoligonucleotides with RecA in the presence of 20 mM Mg²⁺ and 1 mM ATPγS(5-10 μg of DNA in 50 μl of coating buffer). The RecA coating reactionis performed as follows. DNA probes were coated with RecA protein in 10mM Tris-acetate reaction buffer pH 7.5 at 37° C. RecA concentrationduring probe coating depended on the amount and size of the probe. TheMg acetate concentration was 2-20 mM and ATPγS (Pharmacia) was 0.49-4.8mM. The resulting recA-DNA filaments are diluted in 20 mM HEPES bufferand 30 μg DOTAP N- 1-2,3-Dioleoyloxy)propyl!-N,N,N-trimethylammoniummethylsulfate (Boehringer Mannheim) added in 100 μl of 20 mM HEPESbuffer, incubated for 15 min at room temperature, and the resultingmixture added to the cells. The cells are incubated for 5 to 16 h. β-galplasmid is used as a control of the transfection efficiency. ThepSVβ-Galactosidase Control Vector (Promega) was used as a positivecontrol for monitoring transfection efficiencty of Y1 cells. In thisvector the SV40 early promoter and enhancer unit drives transcription ofthe 1acZ gene which encodes the β-galactosidase enzyme. The pSV-β-Galvector was co-transfected together with RecA coated DNA. Standard insitu β-Gal assay (Promega) was performed to determine transfectionefficiency. The observed transfection efficiency is in excess of 90%.Cells are irradiated by light to crosslink coumarin. Changes inmorphology and significant growth inhibition of the Y1 cells is observedupon treatment with the above-described nucleoproteins at a DNAconcentration of 50 nM and higher.

II. Inhibition of episomal replication of the SV40 origin.

COS-7 cells (Gluzman (1981) Cell 23:175-182) are used, which arecharacterized by having an integrated expression cassette providingconstitutive expression of the large T antigen of SV40 with the SV40early promoter. Upon transfection into COS cells, plasmids containingthe SV40 replication origin replicate efficiently in the nucleus asepisomes.

In the following experiments for replication inhibition, the plasmidpSV2SPORT-1 (Life Technologies Mol. Cell. Biol. 2:1044-1051) isemployed. This shuttle vector carries an SV40 origin, SV40 major earlyand late promoters and the transcriptional enhancer. In the experiments,several sequences are targeted in relation to the replication origin.

Plasmid pSV2SPORT-1 (Life Technologies) is transfected into COS-7 cellsby the DEAE dextran method (McCutchan et al. (1968) Natl. Cancer Inst.Monogr.41:351-355) or electroporation. The nucleic acid sequences whichare employed have the following sequences:

pair #1 (45bp)

SEQ ID NO:1! 5'-CAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGT-3'

SEQ ID NO:2! 3'-GTCAATCCCACACCTTTCAGGGGTCCGAGGGGTCGTCCGTCTTCA-5'

pair #2 (45bp)

SEQ ID NO:3! 5'-GAGTCGTATTATAAGCTAGCTTGGGATCTTTGTGAAGGAACCTTA-3'

SEQ ID NO:4! 3'-CTCAGCATAATATTCGATCGAACCCTAGAAACACTTCCTTGGAAT-5'

Nucleoprotein filaments were prepared from these sequences as describedabove and introduced into the cells by lipofection as described above.Cells are irradiated by light to crosslink coumarin. The replicationefficiency is then analyzed by first isolating autonomously replicatingDNA by the standard Hirt procedure (Hirt (1967) J. Mol. Biol.26:365-369). To distinguish between replicated and non-replicatedepisomal DNA, the methylation sensitive restriction enzyme DpnI (Pedenet al. (1980)Science 209:1392-1396) is employed. DpnI digests the GATCsite only when the ⁶ N-position of adenine is methylated on both DNAstrands. The DNA that is used for transfection is cleaved by thisenzyme. Conversely, DNA replicated at least once in mammalian cellslacking dam methylase will be DpnI resistant. The isolated DNA isdigested with DpnI under conditions suggested by the supplier and theresulting fragments separated and resolved by agarose gelelectrophoresis, blotted on nitrocellulose filters and hybridized withradiolabeled pSVSPORT-1. The amount of DpnI-resistant DNA overDpnI-sensitive DNA indicates the efficiency of episomal DNA replication.The replication of pSVSPORT-1 is inhibited more than 90% when thesequences are:

pair #1 (45bp)

SEQ ID NO:5! 5'-CAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGT-3'

SEQ ID NO:6! 3'-GTCAATCCCACACCTTTCAGGGGTCCGAGGGGTCGTCCGTCTTCA-5'

pair #2 (45bp)

SEQ ID NO:7! 5'-GAGTCGTATTATAAGCTAGCTTGGGATCTTTGTGAAGGAACCTTA-3'

SEQ ID NO:8! 3'-CTCAGCATAATATTCGATCGAACCCTAGAAACACTTCCTTGGAAT-5'

these sequences are for both sites in the replication origin. About 50%inhibition of replication is achieved when the targeted sequence is toeither site of the origin of replication. These results are based on acomparison of the replication level where none of the above sequencesare used or where nucleoprotein filaments comprising unrelatedoligonucleotides are used.

To establish the relationship between termination sites and the targetedsequences, 2-D gel electrophoresis is used (Brinton et al. (1991) J.Biol. Chem. 266:5153-5161). The termination sites within the replicativebubble are located at or near the sites targeted by the subjectnucleoproteins.

It is evident from the above results that the subject method andcompositions provide for a convenient and efficient way for modifyingcells, where transcription can be inhibited. The modification may betemporary or permanent depending upon the nature of the nuclear proteinswhich are employed and the ancillary moieties which can act upon thetarget sequence. In this manner, cells may be modulated in culture andin vivo to allow for investigation of physiological processes,preparation of animal models, and therapeutic treatment of cells havingan undesired phenotype.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 8    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 45 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    #ID NO:1: (xi) SEQUENCE DESCRIPTION: SEQ    #45                AGTC CCCAGGCTCC CCAGCAGGCA GAAGT    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 45 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    #ID NO:2: (xi) SEQUENCE DESCRIPTION: SEQ    #45                GAGC CTGGGGACTT TCCACACCCT AACTG    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 45 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    #ID NO:3: (xi) SEQUENCE DESCRIPTION: SEQ    #45                TAGC TTGGGATCTT TGTGAAGGAA CCTTA    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 45 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    #ID NO:4: (xi) SEQUENCE DESCRIPTION: SEQ    #45                AGAT CCCAAGCTAG CTTATAATAC GACTC    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 45 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    #ID NO:5: (xi) SEQUENCE DESCRIPTION: SEQ    #45                AGTC CCCAGGCTCC CCAGCAGGCA GAAGT    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 45 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    #ID NO:6: (xi) SEQUENCE DESCRIPTION: SEQ    #45                CTCG GACCCCTGAA AGGTGTGGGA TTGAC    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 45 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    #ID NO:7: (xi) SEQUENCE DESCRIPTION: SEQ    #45                TAGC TTGGGATCTT TGTGAAGGAA CCTTA    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 45 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    #ID NO:8: (xi) SEQUENCE DESCRIPTION: SEQ    #45                TCTA GGGTTCGATC GAATATTATG CTGAG    __________________________________________________________________________

What is claimed is:
 1. A method of inhibiting the copying of DNA at atarget sequence in a viable cell, said method comprising:introducinginto said cell a pair of nucleoprotein filaments, where thenucleoprotein filaments each comprise a ssDNA strand coated with arecombinase and comprise a region of DNA homology with each other andwith a target sequence in a gene in said cell; whereby a double D-loopis formed and said copying is inhibited.
 2. A method according to claim1, wherein said target sequence is a gene.
 3. A method according toclaim 2, wherein said target sequence is proximal to or overlapping atranscriptional regulatory sequence.
 4. A method according to claim 1,wherein said region of homology is at least 30 nt in length.
 5. A methodaccording to claim 3, wherein said ssDNA strand is at least 50 nt inlength.
 6. A method according to claim 1, wherein joined to at least oneof said ssDNA strand is a molecule that causes scission of dsDNA.
 7. Amethod according to claim 1, wherein joined to at least one of saidssDNA is a molecule that forms a covalent bond with ssDNA or dsDNA underintracellular conditions.
 8. A method according to claim 1, wherein eachof said pair of nucleoprotein filaments comprises first and secondregions that are substantially contiguous when bound to said targetsequence and wherein said first and second regions are joined by a ssDNAlinker.
 9. A method of inhibiting the transcription of a gene in aviable mammalian cell, said method comprising:introducing into said cella pair of nucleoprotein filaments, wherein the nuclcoprotein filamentscomprise a ssDNA strand of at least 30 nt coated with a recombinase andcomprise a region of DNA complementarity with each other and with atarget sequence in said cell, wherein said target sequence is a sequenceof said gene; whereby a double D-loop is formed and transcription isinhibited.
 10. A method according to claim 9, wherein joined to at leastone of said ssDNA strand is a molecule that causes scission in dsDNA.11. A method according to claim 10, wherein said molecule is a metalchelate.
 12. A method according to claim 9, wherein joined to at leastone of said ssDNA strands is a molecule that forms a covalent bond withdsDNA under intracellular conditions.
 13. A kit comprising twonucleoprotein filaments comprising a ssDNA strand coated with arecombinase and comprising a region of DNA homology with each other andwith a target sequence in a cell wherein at least one of said ssDNAstrands is joined to a molecule capable of causing scission in dsDNA orthat forms a covalent bond under intracellular physiologicalconditions;wherein each of said strands comprise first and secondregions that are joined by a ssDNA linker, whereby said first and secondregions are contiguous when bound to said target sequence.